1. Field of Invention
This application relates to the purification of peptides from colostrum. The invention is particularly concerned with the purification of colostrinine from colostrum.
2. Description of Related Art
Colostrum, or foremilk, is a viscous mammary gland secretion characterized by the presence of many elements needed by newborn mammals to develop properly. It is the first lacteal secretion post parturition and contains a high concentration of immunoglobulins (IgG, IgM and IgA) and non-specific proteins. It is replaced by mature breast milk about four to five days after birth. Compared with mature breast milk, colostrum has low sugar content, but is richer in lipids, proteins, mineral salts and immunoglobulins. It also contains various floating cells such as granular and stromal cells, neutrophils, monocyte/macrophages and lymphocytes. It is also rich in growth factors, hormones and cytokines.
Amongst the proteins present in colostrum, caseins are the most prevalent and known to form aggregates (micelles), which are similar in all mammals. Many proteins and peptides are bound to those aggregates, by weak hydrophobic and ionic forces. The resultant network of proteinaceous micelles has the ability to trap many small molecular weight compounds of differing nature, such as lipids, carbohydrates, and peptides, forming a unique homogeneous solution. The micelles help to distribute these micro-molecules relatively uniformly throughout the colostrum, and also prevent them from the formation of unwanted aggregates.
A number of peptides from milk with various biological activities have been reported. Some peptides exist naturally and some can be released via enzymatic proteolysis of the parent milk proteins. Of particular interest are those naturally existing peptides that are bound to casein micelles. Besides casein protein, calcium and phosphate, the micelle also contains citrate, minor ions, lipase and plasmin enzymes, and various peptides entrapped in their structure. Therefore, the downstream purification processing of many milk-derived components recently become the most challenging effort for the dairy industry.
In general, the downstream processing of milk starts by coagulating caseins with addition of chymosin to give curds, which are then separated from the liquid, whey, after which they can be processed and matured to produce a variety of cheeses. Chymosin brakes down the kappa-casein molecules causing a global collapse of casein micelles. As a result many components of a soluble fraction of milk become entrapped in the precipitate and disregarded. Particularly small molecular peptides with a high hydrophobic index, such as colostrinine, can be lost.
The original purification protocol for obtaining colostrinine (Janusz et al.), consists of pH-dependent casein precipitation followed by various chromatographic steps, including ion exchange, affinity and molecular sieving, combined with ammonium sulfate precipitation. Although, this method is reproducible, it is laborious and difficult to scale-up for the industrial applications. Since then numerous protocols, utilizing membrane filtration, have been developed for recovery of low molecular weight peptides from milk and colostrum, but all of these have had shortcomings.
Tangential flow filtration is used in one standard protocol for separating milk components in the dairy industry. For example, in the U.S. Pat. No. 6,268,487 entitled Purification of Biologically Active Peptides from Milk by Kutzko et al., a method for separation of milk components by tangential flow filtration is disclosed. Also, an ultrafiltration is proposed by Roger et al., in a U.S. Pat. No. 4,485,040, entitled: Process for Obtaining an α-Lactalbumin Enriched Product from Whey, and Uses Thereof, to separate milk components on a membrane having cut-off greater than 5,000 (e.g. 50,000) at a pH between 6.3 and 7 (e.g. 6.6) and a temperature between 30° C. and 60° C.
The use of ultrafiltration is also described in U.S. Pat. No. 4,816,563 Wilson et al., entitled: Process for Obtaining Transfer Factor from Colostrum, Transfer Factor so Obtained and Use Thereof. In fact, filtration of milk or colostrum is a standard procedure in the dairy industry today. Wilson et al., describes the use of an agent to prepare a transfer factor from colostrum. Suitable agents are said to include alcohols, ketones and polyethylene glycol. There is, however, no description of the recovery of colostrinine from colostrum.
U.S. Pat. No. 5,216,129 discloses a process for obtaining kappa-caseino-glycomacropeptide from a whey product concentrated in proteins. The process involves the use of ethanol at a concentration of 5% to 25% based on the volume of the solution. The whey product is not colostrinine.
UK Patent 1,438,008 discloses a process for the extraction of a specific octapeptide from frog skin using alcohol.
JP520062796 discloses a method of extracting cyclic peptides from the root of Ribia akane Nakai using, inter alia, an alcohol.
In J Pharm Pharmaceut Sci, Vol 5, 2002. MEC Lutsiak et al., “Analysis of peptide and lipopeptide content in liposomes”, p 279-284, there is disclosed a method for extracting peptides from liposomes.
In Journal of Antibiotics, Vol XL, 1987, E Meyers et al, “Xylocandin: a new complex of ant/fungal peptides I. Taxonomy, isolation and biological activity”, p 1515-1519, there is disclosed method of extracting peptides from the bacteria pseudomonas cepacia. 
In Acta Endocrinologica, vol 111, 1986, WF Blum et al, “Isolation and partial characterisation of six somatomedin-like peptides from human plasma Cohn fraction IV”, p 271-284, there is disclosed the extraction of somatomedin-like peptides from human plasma using ethanol.
In J Dairy Research, vol 54, 1987, DS Home, “Ethanol stability of Casein micelles—a hypothesis concerning the role of calcium phosphate”, p 389-395, there is disclosed a hypothetical elaboration on casein micelles structure and calcium release.
In Ir. J. Fd. Sci. Techno!., vol 9, 1985, M M Hewedi et al, “Recovery of milk protein by ethanol precipitation”, p 11-13, there is disclosed a method of precipitating milk proteins using ethanol.
COLOSTRININ®, also known as colostrinine, proline-rich polypeptide or PRP, was first isolated in 1974 (Janusz et al, FEBS Lett., 49, 276-279) from ovine colostrum. Certain therapeutic uses of COLOSTRININ®, particularly in the treatment of Alzheimer's disease, were described in WO98/14473, the contents of which are incorporated herein by reference. In this patent application, the physical characteristics of colostrinine, as deter inable at the time, were described. Although the physical characteristics were correct, the understanding of colostrinine has moved on since this application was filed. WO98/14473 also described a method for extracting colostrinine from raw colostrum, which is often referred to as the “Janusz” method. This method is presently the principle method of extracting colostrinine from colostrum. It has the disadvantages that the industrial scale up is difficult to obtain and yields from the method are low.
WO00/75173, the contents of which are incorporated herein by reference, describes a number of peptides found in colostrinine. WO02/46211, the contents of which are incorporated herein by reference, describes a number of other peptides which can be found in colostrinine.
Of considerable interest in colostrinine is the presence of various polypeptides that can be isolated only from colostrum, not mature milk. During the days following parturition, the concentration of colostrinine in a mammary gland secretion precipitously diminishes through the end of the third day after delivery. Such a short lifetime for some of the colostrinine peptides indicates their important role in early development of infant's immune system and the protection of newborns against environmental shocks.
Colostrinine has more recently been discovered to exist in two forms; free and aggregated (bound). It is thought that free forms of colostrinine are required to protect the newborn mammal from oxidative stress, which appears immediately after birth. The bound or aggregated forms are designed to maintain this function for prolonged periods of time after birth. The bound form of colostrinine, in addition, is thought to participate in the development and/or protection of different organs and systems. This takes place when oxidative stress problems subside, and free colostrinine concentrations begin to decrease. The bound form of colostrinine is slowly released into the body fluids to modulate physiological functions when the free form is exhausted. This model finds support in studies on the gradual disappearance of specific colostrinine peptides from colostrum after parturition.
The colostrinine complex is now believed to consist of at least five subgroups of peptides; each subgroup has its own characteristic hydrophobic pattern. Evidence suggests that these peptides have a tendency to form aggregates, due to the presence of specially arranged non-polar, polar, aromatic, positively- and negatively-charged amino acids. Furthermore, the amino acid compositions of the peptides and their hydrophobic character further suggest this aggregating ability.
From the presently available information, we have found that colostrinine peptides have the best biological activity when they are present in their native form. When they are purified, they start to interact with each other forming non-covalently bound complexes with apparently weaker biological activities. It is believed that colostrinine is a mixture of more than 62 separate peptides, derived from precursor proteins, such as annexin, beta-casein, a hypothetical beta-casein homologue and others with no homology to any specific protein in the current GenBank database.